The present invention relates to a method for recovering recombinant HBsAg from HBsAg expressing yeast cells.
The destruction of the cell wall of a microorganismxe2x80x94cell disruptionxe2x80x94is the first step in recovering intracellular, biologically active proteins. On a production scale, mechanical disruption methods, such as wet milling in ball mills, have turned out to be suited in recent years.
The high-speed agitator ball mills of a closed constructional type have developed from the conventional ball mills used in pigment processing because of the continuously increasing demands made on comminution and dispersion. They consist of a hollow cylinder rotating about a vertical or horizontal axis, which is filled with mixed oxide beads of glass or zirconium up to a filling degree of 80%. The rotational speed can here not be increased in any desired way for increasing impact/pressure crushing because the centrifugal forces compensate the grinding effect. Furthermore, at increased speeds or filling degrees thermal problems arise that might damage the product to be recovered. A continuous grinding material/grinding body separation is carried out with a sieve cartridge, a rotating sieve gap or with a coaxial annular gap integrated into the bearing housing. The grinding material is cooled via a double jacket around the grinding container; the rotating shaft can also be cooled in part. The disruption principle of wet milling consists in transmitting motional energy from the agitator to the grinding body. This is predominantly done by adhesion forces in combination with displacement forces that are due to the assembly and type of agitator elements. The grinding chamber is activated by this and by cohesive forces. The acceleration of the grinding body in radial direction effects the formation of a laminar flow. Depending on the absolute speed and the size of the grinding bodies moved, the differential speed profiles between the grinding body layers effect high shear forces which apart from collision results are mainly responsible for the destruction of the cell wall of the microorganism.
Cells walls can also be broken mechanically by high pressure homogenizers. Such a device consists essentially of a high-pressure piston pump and a homogenizing unit. A homogenization valve will open when the set pressure is reached, the cell suspension being then pressed through the valve unit at a very high speed. The cell suspension is thus heated by about 2.5xc2x0 C. per 100 bar. After having left the disruption valve unit the cell suspension is cooled by means of a heat exchanger. Thus, in contrast to wet milling, the disruption material is here heated for a short period of time. While passing through the homogenizing unit the cell suspension is subjected to very high turbulence, cavitation and shear forces. The disruption principle of a high pressure homogenizer consists in the sudden reduction of the energy density in the cell suspension within an extremely short period of time, i.e. the high pressure difference and the rapid pressure drop can be regarded as the main factor for the degree of disruption.
Both mechanical disruption methods are used for different microorganisms. The selection of the respective method depends on the type of the microorganism, in particular on its structure. For instance, both cell disintegration methods are tested in Pittroff, M. et al., DECHEMA-Biotechnol. Conf.; (1990), 4, Pt.B, 1055-60 for various microorganisms, such as Saccharomyces cerevisiae, Micrococcus luteus and Escherichia coli, and the conclusion could be drawn that morphological differences in the microorganism affect the disintegration performance of the two methods.
For instance, Pittroff, M. et al., DECHEMA-Biotechnol. Conf.; (1992, 5, Pt.B, 687-91, and Pittroff, M. et al, Chem. Ing. Techn.; (1992), 64, 10, 950-53 point out that the high-pressure disruption method is preferably carried out for specific microorganisms, such as rod-shaped bacteria. By contrast, the ball milling method is preferably used for cocci having a round structure. The same disintegration degree was observed for both methods in the disruption of yeast cells of the species Saccgarintces cerevisiae. 
Furthermore, Luther, H. et al., Actaxe2x80x94Biotechnol.; (1992), 12, 4, 281-91, describe a further comparison between ball mills and high pressure homogenizers for purifying proteins from Saccgarintces cerevisiae or Escherichia coli. It is confirmed that both yeast and bacterial cells can be disrupted using ball mills or high pressure homogenizers. It has been found to be a disadvantage of high pressure homogenizers that slime-forming microorganisms or mechanical contamination very easily lead to occlusions. It is pointed out with respect to yeast cells that the high pressure homogenizer destroys fewer cells. Although this has not been mentioned explicitly, a lower yield from the high pressure homogenizer has to be assumed.
Schxc3xctte, H., Biol. Recombinant-Microorg. Anim. Cells; (1991) Oholo 34 Meet., 293-305 also confirm that high pressure homogenizers effect an efficient disruption of yeast and bacterial cells, but are not very suitable for mycelial organisms.
Saccgarintces cerevisiae disruption using a high pressure homogenizer was studied in Baldwin, C., Biotechnol. Tech; (1990), 96, 4, 329-34. It was found that the breakage of cells by means of a high pressure homogenizer generally gave low disruption yields (40% in 5 passes). A total disruption of yeast cells in the high pressure homogenizer could only be observed if an enzyme treatment with zymolase from Oerskovia xanthineolytica had been performed previously.
As becomes further apparent from the extensive studies that have so far been carried out, the type of the protein to be purified is of decisive importance to the selection of the respective disruption method. Of particular interest is here the cell disruption of hepatitis B surface antigen (HBsAg)-expressing cells.
Choo, K. B. et al., Biochem. Biophys. Res. Commun.; (1985), 131, 1, 160-66 study the use of ball mills for the disruption of Saccgarintces cerevisiae cells for the recovery of HBsAg. However, only a low amount of HBsAg could be extracted in particulate form.
Fenton D. M. et al., Abstr. Ann. Med. Am. Soc. Microbiol.; (1984), 84 Meet. 193 describe the release of recombinant HBsAg from Saccgarintces cerevisiae by means of cell disruption in a high pressure homogenizer. It is pointed out in this document that the solubilization of HBsAg in an active, i.e. antigenic, form on a large scale poses problems. Sufficient cell breakage and satisfactory protein release were only observed in a high pressure homogenizer after ten passes, and a maximum HBsAg release could only be observed after 15 passes. The authors drew the conclusion that the release of antigenically active HBsAg requires the disruption of subcellular structures.
Hence, up to now it has not been possible with the two disruption methods to provide suitable systems for the optimum production of HBsAg by cell disruption.
It is therefore the object of the present invention to develop a method for recovering a high yield of recombinant HBsAg from recombinant microorganisms.
According to the invention this object is achieved by a method for recovering recombinant HBsAg, wherein recombinant methylotrophic yeast cells which are capable of expressing HBsAg are disrupted using a high pressure homogenizer, and HBsAg is recovered from the cell debris obtained.
Surprisingly enough, it has been found that in the cell disruption of HBsAg-expressing Hansenula polymorpha cells in a high pressure homogenizer a considerably higher product yield per g of cell dry weight could be achieved than with the conventional methods, in particular the cell disruption of Saccharomyces cerevisiae by means of a high pressure homogenizer or glass bead mills. The method of the invention is thus a considerable improvement over the formerly known methods used for recovering HBsAg from microorganisms.
In the method of the invention, recombinant HBsAg is recovered from recombinant methylotrophic yeast cells which are capable of expressing HBsAg. Preferably, an HBsAg-expressing strain of the species Hansenula is used, particularly preferably Hansenula polymorpha. Methylotrophic yeasts of the species Pichia, Candida and Torulopsis can also be used. During fermentation the standard parameters, such as pH, aeration and temperature, are controlled. Glycerol, methanol and glucose, preferably glycerol, are suited as the substrate. The substrate is supplied to the fermenter culture until the yeast cells reach a desired cell density of at least 80, preferably at least 90 gxc2x7lxe2x88x921 cell dry weight. After said cell density has been reached, the expression of HBsAg is induced in the yeast culture.
In preferred embodiments the expression of HBsAg in methylotrophic yeast is controlled by a promoter which derives from a gene involved in methanol metabolism. Well-described promoters are the MOX promoter, the DAS and the FMD promoter (Ledeboer, A. M. et al., Nucl. Acids Res. (1985), 13, 9, 3063-3082; Janowics, Z. A. et al., Nucl. Acids Res. (1995), 13, 9, 2043-3062, EP 299 108). To achieve an optimum expression under the control of said promoters, the yeast cells are first grown in a fully synthetic nutrient medium. Methanol is added for induction so that the cell suspension has about 1% of methanol. Detailed information on the culturing of methylotrophic yeasts and the induction conditions for the three above-mentioned promoters can e.g. be found in Gelissen, G. in Murooka/Imanaka (eds.) Recombinant microbes for industrial and agricultural applications, Marcel Dekker, NY 1993, 787-796 and Weydemann, U. et al., Appl. Microbiol. Biotechnol. (1995), 44, 377-385.
Upon completion of the fermentation process the cells are separated from the medium components via tangential flow filtration. A desired cell density is adjusted by suitable dilution with a disruption buffer.
For disruption the yeast cells are preferably used at a cell density of 50 to 150 gxc2x7lxe2x88x921 cell dry weight. The exact cell density depends on the respective recombinant methylotrophic yeast cell species. In particular, a cell density of 70 to 120 gxc2x7lxe2x88x921 cell dry weight is preferred. If the cell density of the recombinant methylotrophic yeast cells to be used according to the invention is too low, the method becomes time-consuming and uneconomic. At an excessively high cell density of the yeast cells, the disruption becomes more and more inefficient, and the demands on the cooling capacity are increasing.
The high pressure homogenizer used according to the invention is a conventional high-pressure homogenizer, as described in the introduction, which can be used for cell disruption. Particularly preferred is the use of a high pressure homogenizer of the type Nanojet LAB30 PL or a comparable model. During cell disruption the pressure in the homogenizing unit is 1000 to 2000 bar. A pressure of 1200-1600 bar is preferred. Particularly preferred is a pressure of 1500-1600 bar.
The starting temperature of the yeast cells is preferably 2-15xc2x0 C., particularly preferably 48-8xc2x0 C. The yeast cells which are normally present in the form of a cell suspension can be cooled under stirring to the desired temperature. The high pressure homogenizer is preferably cooled, so that a low outlet temperature of e.g. 2-15xc2x0 C., preferably 3-13xc2x0 C., particularly preferably 5-10xc2x0 C., is observed at the product outlet.
In the high pressure homogenizer the cells are normally disrupted in several cycles (passes). It has been found that a total of 3 to 8 cycles (passes) are sufficient for an optimum product yield. Preferred are 3 to 6, particularly preferred 4 cycles (passes) for the cell disruption for recovering recombinant HBsAg.
After completion of the cell disrupting process the preparation produced in this way is preferably subjected to further separating and purifying steps. Said additional process steps are conventional methods which effect a further purification and enrichment of recombinant HBsAg in the extract. For instance, one or more of the following separating and purifying steps can be carried out, e.g. in the order indicated:
Precipitation of the cell debris with polyethylene glycol, separation of the PEG supernatant, adsorption on a silica matrix, separation of the silica matrix, desorption of the product from the silica matrix, separation of the supernatant of the silica matrix, ion exchange chromatography, concentration of the ion exchanger pool by ultrafiltration, density gradient separation in cesium chloride, size-exclusion chromatography and sterile filtration (final aqueous bulk).
The following example will explain the invention.